2.4. HPLC Analysis of Carotenoids

The procedures for the isolation and identification of carotenoid pigments and its esters have already been described in previous works [11,12]. Quantitative analysis of carotenoids was carried out by HPLC according to Atienza et al. [11]. The HPLC system consisted of a Waters e2695 Alliance chromatograph fitted with a Waters 2998 photodiode array detector, and controlled with Empower2 software (Waters Cromatografía, S.A., Barcelona, Spain). A reversed-phase column (Mediterranea SEA18, 3 μm, 20 × 0.46 cm; Teknokroma, Barcelona, Spain) was used. Separation was achieved by a binary-gradient elution using an initial composition of 75% acetone and 25% deionized water, which was increased linearly to 95% acetone in 10 min, then raised to 100% in 2 min, and maintained constant for 10 min. Initial conditions were reached in 5 min. An injection volume of 10 μL and a flow rate of 1 mL/min were used. Detection was performed at 450 nm, and the UV-visible spectra were acquired online (350–700 nm wavelength range). Quantification was carried out using calibration curves prepared with lutein, zeaxanthin and β-carotene standards isolated and purified from natural sources [27]. Calibration curves including eight-points were prepared in the pigment concentration range of 0.5–45 μg/mL. Lutein ester content were estimated by using the calibration curve for free lutein, since the esterification of xanthophylls with fatty acids does not modify the chromophore properties [28]. Accordingly, the concentration of lutein esters was expressed as free lutein equivalents. The calibration curve of free lutein was also used to determine the concentration of the (Z)-isomers of lutein. Data were expressed as μg/g dry weight (μg/g dw).